1. Field of the Invention
The present invention relates to an improved analytical element for quantitative analysis of substance contained in aqueous liquids, and particularly contained in body fluids.
2. Description of Prior Arts
A variety of methods for analysis of substance contained in water, foodstuffs and body fluids are heretofore proposed. Particularly a method comprising procedures of promoting formation of colored material in a liquid sample which occurs in proportion to the amount of the substance under analysis (analyte) and measuring the color density is well known in the analysis. This method has been utilized not only in solution analysis (wet analysis) but also in dry analysis. Such dry analysis utilizes, for instance, a dry sheet similar to a pH test strip wherein a paper or other absorbent carrier is impregnated with a reagent for forming color upon contact with an analyte.
Also known is an analytical element sheet or film having single-layer or multi-layer structure for the dry analysis, which is appropriate for quantitative analysis of analyte with high accuracy.
The above-mentioned multilayer analytical element is also termed a multilayer analytical material, and various elements are known. For instance, a multilayer analytical element comprising a support, one or more reagent layers provided thereon and a non-fibrous porous spreading layer provided thereon is disclosed in U.S. Pat. Nos. 3,630,957, 3,992,158, 3,983,005, 4,042,335, 4,069,017, and 4,144,306, and U.S. Pat. Re. No. 30,267, and others.
In the multilayer analytical element, when applied an aqueous liquid sample containing an analyte onto the spreading layer, the aqueous liquid sample permeates the reagent layer maintaining a uniform amount per unit area to produce color formation reaction. After a certain period of time, the color density is measured to determine a concentration analyte in the aqueous liquid sample.
Representative examples of the analytes analyzable by means of the above-described multilayer analytical element include a variety of hydrolase contained in body fluids, such as, blood, urine, intestinal juice, saliva, spinal fluid and pancreatic juice. In addition to the hydrolase, other substances contained in body fluids, such as, cholesterol, triglyceride (neutral fat), glucose and uric acid, which are decomposable by action of oxidoreductase, especially oxidase, to produce a low molecular weight compound and hydrogen peroxide, can be mentioned.
At present, various hydrolases are known. The multilayer analytical element is particularly useful for analysis of hydrolase whose substrate is a high molecular weight compound and which produces a diffusive low molecular weight product through hydrolysis. Quantitative analyses of the hydrolase is important in clinical test, and a simple and accurate analytical method is highly desired. Examples of such hydrolase include amylase, lipase, protease such as trypsin, chymotrypsin and pepsin, pectinase and various kinases.
For instance, measurement of amount of amylase contained in blood is clinically very important for observing conditions of pancreas function. Amylase is one of hydrolases that hydrolyzes amylose linkages of starch or the like and produces a low molecular weight polysaccharide, oligosaccharide or monosaccharide. Amylase is generally analyzed by a method of measuring the enzymic activity and calculating a relative value of its concentration in the liquid sample.
The aforementioned Japanese patent provisional publication No. 53(1978)-131089 describes a dry multilayer analytical element capable of measuring the amylase activity by means of an extremely simple procedure. The multilayer analytical element has a reagent layer comprising a substrate of amylase such as a non-diffusive substrate carrying a detectable chromophore (e.g. starch carrying dye groups), and a registration layer for receiving a diffusive reaction product given by hydrolyzing action of analyte, that is, amylase. In the method of analysis of the amylase activity employing this multilayer analytical element, a non-diffusive substrate carrying a detectable chromophore (dye or the like) is decomposed by action of the amylase contained in a liquid sample to produce a diffusive low molecular weight product having chromophore. The low molecular weight product is diffused in the reagent layer, and finally received and fixed in the registration layer. Then, the amount of the reaction product can be determined by measuring photometrically the color density proportional to the amount of the diffusive low molecular weight product carrying chromophore received by the registration layer.
The aforementioned Japanese patent provisional publication No. 51(1976)-40191 discloses an analytical element having a layer constitution similar to that described in the above-mentioned Japanese patent provisional publication No. 53(1978)-1310899. The former involves procedures of converting originally undetectable chemical species to diffusive and detectable chemical species in a reagent layer under action of analyte, and detecting the chemical species in a detection layer (registration layer), while the latter element contains originally detectable chemical species in a reagent layer.
In these arts, a detection layer or registration layer functions only for receiving a diffusive chemical species, and no color formation reaction takes place therein. Accordingly, in order to clearly distinguish the detectable chemical species remaining in the reagent layer from the chemical species received in the detection layer or registration layer and to obtain quantitative analytical results, a radiation blocking layer having light blocking or shielding characteristics is necessarily provided therebetween for the purpose of color blocking.
Japanese patent provisional publication No. 57(1982)-40649 describes a multilayer analytical element improved in the above-described defect. In the multilayer analytical element, a substrate contained in a substrate layer of the element reacts with analyte to give a substantially colorless diffusive product, and the diffusive product then reacts with a chromogen to form a detectable color. Generally, the chromogen is previously introduced into the multilayer analytical elment in such manner that the chromogen is contained in a color reaction layer attached to the substrate layer. Accordingly, by the use of this multilayer analytical element, the reaction product is easily distinguished from the unreacted substrate.
However, in the case that a diazonium compound is employed in the last-described art as the chromogen which reacts with a diffusive product to form a detectable color, the diazonium compound is unstable and undergoes decomposition reaction and various undesirable side reactions, whereby giving poor quantitative results.
Generally, the diazonium compound is known to be unstable. However, it is also known that the above-described decomposition reaction or various side reactions are reduced by introducing an acid such as citric acid, tartaric acid, phosphoric acid or naphthalenesulfonic acid into a reaction system to lower pH for keeping the system in acidic conditions. In addition, it is further known that an anion originating from zinc chloride, tetrafluoroborate, hexafluoroaluminate and the like can be present as a counter ion of the diazonium compound to stabilize the diazonium compound. Those arts are described in "PHOTOSENSITIVE DIAZO COMPOUND" by M. S. Dinaburg (THE FOCAL PRESS, 1964). In the art, although the diazonium compound is stabilized by the adjustment of pH of reaction system, the so adjusted pH value unfavorably deviates from the optimum pH range of analyte (for instance, around pH 6.9, such as pH 6.5-7.5, for human amylase). Accordingly, the enzymic activity of the analyte does not appropriately function under these conditions, and no reliable analytical result is obtained.
The above-described problem on the stabilization of the diazonium compound has been solved by employing a polycarboxylic acid as binder. It has been then discovered that the stabilization of a diazonium compound is accomplished without disturbing the analytical reaction involved. This invention has been applied for patent in Japanese patent application No. 57(1983)-140736.